A fundamental characteristic of normal cells is their limited ability to proliferate in culture. While differentiated cells may have potential in vivo proliferative capacity, once placed in culture, the cells typically undergo an initial milotic period that is invariably followed by a gradual decline in cell division. This decrease in cell division is virtually irreversible and complete. Cell death usually occurs, although the cells may remain viable for long time periods. This progression of events from actively dividing cells to nondividing cells is termed "cellular senescence" or the "finite lifespan phenotype."
Only some primitive stem cells (e.g., the inner cell mass of early embryos) may have the potential for unlimited proliferation in culture. In vivo, differentiation serves to restrict stem cell proliferation during organismal development. There are several lines of evidence which indicate that cells senesce in vivo, due to the finite lifespan phenotype.
Cell senescence was first systematically described approximately 30 years ago, in such publications as L. Hayflick, "The limited in vitro lifetime of human diploid cell strains," Exp. Cell Res., 37:614 (1965); and Hayflick and Moorhead, "The serial cultivation of human diploid cell strains," Exp. Cell. Res., 25:585 (1961). Despite the fact that cell senescence was first described long ago, our molecular understanding of cell senescence is still incomplete. However, the limited in vivo data available indicate that senescence is not merely an artefact of cell culture. Rather, it appears to be genetically-based. Largely due to the lack of an animal model or useful in vivo methods to permit its study in humans and other animals, cell cultures are the most common method used to study the processes associated with cell senescence.
Various agents, such as carcinogens (e.g., chemicals, viruses and oncogenes) are capable of immortalizing normal cells. By itself, immortality is insufficient for neoplastic transformation. However, most immortal cells have an increased tendency for spontaneous, carcinogen-induced, or oncogene-induced neoplastic progression. Thus, escape from senescence may be a pre-neoplastic change that results in the predisposition to neoplastic conversion. Based on these observations, it has been hypothesized that cellular senescence is a major mechanism involved in tumor suppression. O'Brien et al., "Suppression of tumor growth by senescence in virally transformed human fibroblasts," Proc. Natl. Acad. Sci., 83:8659 (1986).
Of the various theories used to explain senescence, there is much evidence to indicate that senescence is a genetically programmed process. Based on cell fusion studies (e.g., microcell fusions to produce hybrid cells), it has been determined that at least four genetic complementation groups are associated with immortality. Smith and Pereira-Smith, "Genetic and molecular studies of cellular immortalization," Adv. Cancer Res., 54:63 (1990); Pereira-Smith and Smith, "Genetic analysis of indefinite division in human cells: Identification of four complementation groups," Proc. Natl. Acad. Sci., 85:6042 (1988); Smith and Pereira-Smith, "Altered gene expression during cellular aging," Genome 31:386 (1989); and Pereira-Smith and Smith, "Evidence for the recessive nature of cellular immortality" Science 221:964 (1983).
Individual chromosomes have been associated with the induction of senescence. For example, human chromosome 1 was shown to induce senescence in such cells as an immortal hamster cell line, while chromosome 11 had no effect on the growth of the cells [Sugawara et al., "Induction of cellular senescence in immortalized cells by human chromosome 1," Science 24:707 (1990); Ning et al., "Tumor suppression by chromosome 11 is not due to cellular senescence, Exp. Cell Res., 192:220 (1991); and Ning and Pereira-Smith, "Molecular genetic approaches to the study of cellular senescence," Mut. Res., 256:303 (1991).] Human chromosome 4 was found to limit the proliferative lifespan of three immortal human tumor cell lines [Ning et al., "Genetic analysis of indefinite division in human cells: Evidence for a cell senescence-related gene(s) on human chromosome 4," Proc. Natl. Acad. Sci., 88:5635 (1991)]. Thus, at least two different human chromosomes have been shown to reverse the immortal phenotype following their introduction into established cell lines. See also, Wright et al., "Reversible cellular senescence: Implications for immortalization of normal human diploid fibroblasts," Mol. Cell. Biol., 9:3088 (1989).
Furthermore, there is evidence that oncogenes can immortalize human cells, usually by encoding multifunctional nuclear proteins for which there is neither an identified single structural cellular homologue, nor an identified single function cellular homologue. These proteins bind and may serve to inactivate at least two tumor suppressor gene products (e.g., the cellular proteins p53 and the retinoblastoma susceptibility gene product [Rb])[McCormick and Campisi, "Cellular aging and senescence," Current Opinion in Cell Biology 3:230 (1991)]. These and other genetic experiments indicate that senescence is dominant over immortality [see e.g., Pereira-Smith et al., "Immortal phenotype of the HeLa variant D98 is recessive in hybrids formed with normal human fibroblasts," J. Cell. Physiol., 142:222 (1990)]. Indeed, it is hypothesized that multiple genes might be lost or inactivated during cellular escape from senescence.
Recent investigations indicate that the cessation of cell division is just one facet of a very complex senescent phenotype. For example, changes in cell function are also associated with cell senescence. Further complexity is added by studies on cultured well-differentiated epithelial cells which suggest that changes in differentiated functions of cells and the arrest of cell proliferation can be dissociated from each other [Yang and Homsby, "Dissociation of senescence-associated changes in differentiated gene expression and replicative senescence in cultured adrenocortical cells," J. Cell Sci., 94:757 (1989)]. Thus, in order to gain a full understanding of the nature and significance of cell senescence, an understanding of both types of changes (i.e., those that result in growth inhibition and those that alter differentiated functions) must be gained.
Attempts to gain this understanding are hampered by the conditions and testing methods in current use. There are relatively few assays that can distinguish normal and senescent cells. For example, unlike normal cells, cultured senescent cells do not proliferate in response to milogens. In addition, there is no method to distinguish senescent cells from quiescent, terminally differentiated or physiologically compromised cells in tissues without culturing the cells that migrate out of tissue explants. These studies require cumbersome, labor and time-intensive methods which are not suited to rapid and cost-effective assessment of the status of cells in vivo or in vitro. Furthermore, these assays are not definitive nor predictive in terms of the actual physiological characteristics of the cells.
Thus, the majority of the present assay systems rely on assessment of gene expression, such as methods involving incorporation of radiolabelled thymidine or thymidine analogues. In these assays, senescent cells do not incorporate the radiolabelled compound. However, quiescent cells also do not incorporate these compounds. Therefore, this type of assay is only useful under carefully controlled culture conditions. Importantly, most of these methods are toxic to cells, limiting their value if continuing cell growth is desired or required for further study. In addition, these toxic compounds present concerns regarding the cost and safety of their disposal. Furthermore, they cannot be used to identify senescent cells present in heterogeneous cell cultures.
What is need is a rapid, convenient, easy-to-use, means to identify senescent cells present in a mixed cell population. Such a method should ideally permit the researcher to differentiate senescent from quiescent, terminally differentiated or physiologically compromised cells.